Shouting at the ocean with pebbles in my mouth since 2008. The subjects of this blog include forensics, the war-on-terror detainees, the Duke lacrosse case, the Knox/Sollecito case, and the academic world as it intersects the political. It will sometimes examine issues of particular interest to Wilmington, NC and the University of North Carolina at Wilmington.

Thursday, September 22, 2011

Amanda Knox and Raffaele Sollecito: There is no mixed blood

Part 32 in the Knox/Sollecito case

IntroductionThe Conti-Vecchiotti report casts further doubt upon the reliability of the bra clasp and the kitchen knife, two of the strongest, yet seriously flawed, pieces of evidence against Ms. Knox and Mr. Sollecito in this case. With the appeals trial expect to wrap up shortly, it is time to reexamine the strength of the other biological evidence against them, especially the mixed DNA traces. An anonymous pro-guilt blogger did so several months ago. The related question of whether or not the luminol-positive areas (some of which had DNA profiles) were truly blood was the subject of a previous entry here and a discussion at Let’s Talk about True Crime.

Which samples contained mixed DNA?There are two amorphous, luminol-positive spots in Filomena’s room (Reps. 176 and 177), although the alleles which may be Amanda’s are quite weak. There is a shoe print in the hallway (Rep. 183). In the shared bathroom there is a sample from the bidet drain, the cotton flock container on the floor, and from the inside of the washbasin (Rep. 137). There is no DNA from Amanda in the murder room, either by itself or mixed with Meredith’s DNA.

Did the jury in the first trial find the mixed DNA to be significant?Barbie Nadeau wrote, "The defense's biggest mistake, according to interviews with jurors after the trial, was doing nothing to refute the mixed-blood evidence beyond noting that it is common to find mingled DNA when two people live in the same house. The jurors needed more than that. 'To have mixed blood, you have to both be bleeding,' one of them remarked to me after the verdict. It was obvious that Meredith was bleeding, but why was Amanda bleeding?'" ("Angel Face," pages 152-153)

Does mixed DNA indicate mixed blood?The passage quoted above implies that at least one juror thought that the two were equivalent, but this is a fallacy. There are three possibilities with respect to mixed DNA and blood: neither source, one source, or both sources of DNA could be blood. A commonly repeated fallacy of this case is to equate a mixture of DNA with a mixture of blood (to assume possibility three). For example in a recent Dateline broadcast Barbie Nadeau said, “I have to say, I live in a house with 3 people, my two sons and my husband, I guarantee you I have no mixed blood with any of them, anywhere in my house. I don't bleed where they bleed, we don't bleed at the same time. There would never be my mixed blood, their blood and my blood anywhere ever.”

The false equivalence between mixed DNA and mixed blood dates back at least to the publication of the book Darkness Descending. In this book Colonel Luciano Garofano, a retired officer of the Carabinieri, said, “However, here is the electropherogram and you can see that the RFU value is very high, so the sample is undoubtedly blood, which is the body fluid that provides the greatest amount of DNA. In some cases you see higher peaks of Amanda's DNA than Meredith's. Amanda has been bleeding. Nor is it old blood, as the defence might say, because blood decays fast. We have the same result on the cotton-bud box. The light switch was over-scrubbed, but from the film the way the cotton-bud was good enough. There too we have mixed blood. So that's pretty significant for Amanda, Unfortunately for her, she bled at the same time Meredith was bleeding. That's a lot to explain." (Darkness Descending, page 371).

There are several reasons why Garofano’s interpretations are wrong. In response to a question of mine, Professor Dan Krane wrote, “Inferring tissue source from peak heights is just plain silly -- to the point of being absolutely outrageous. It hardly bears more comment than that, but if high peaks mean blood then what would you expect from semen which has a ten to one hundred fold higher concentration of DNA?” Professor Greg Hampikian concurred with the view that peak heights were not an indication of whether or not blood was the source of DNA. The peak heights for Raffaele’s profile on the cigarette butt were reported to me as being about the same height as those on the cotton box, and the former are presumably from saliva.

Colonel Garofano’s claim that the DNA from blood decays quickly is difficult to evaluate. A paper (Park et al., “Direct STR Amplification from Whole Blood and Blood- or Saliva-Spotted FTA without DNA Purification,” J. Forensic Sci., March 2008, Vol. 53, No. 2, 335-41) showed that 1-2 year-old blood samples gave strong signals in DNA profiling when stored in the form of FTA cards (which contain stabilizers); therefore, their study does not exactly refute what Colonel Garofano claimed, but it does not support his claim, either. However, this paper also showed that saliva gave tall peaks in DNA profiling, which is one more indication that peak height cannot be used to infer the biological origin of a sample. The rate of decay of a DNA sample depends upon so many factors that dating DNA by its degradation is not practical. Furthermore, even if one were to accept that DNA peaks from blood did degrade very quickly, one might have to conclude that the luminol-positive, mixed-DNA samples were not blood, inasmuch as the luminol was applied on 18 December, more than a month and a half after the crime.

What did Massei conclude with respect to the mixed DNA samples?In contrast to the juror quoted above, the Massei report did not assume that mixed DNA was equivalent to mixed blood (pp. 278-279, English Translation). “It should then be highlighted that in that same bathroom various [300] trace specimens were found, of a mixed nature and testing positively for blood. It is true that, according to what was asserted and explained, it is not possible with a mixed trace specimen that tested positive for human blood to determine which of the trace’s contributors the blood belongs to. In this case, however, non-mixed traces were also found, which were shown to be of a haematological nature [i.e. blood] and turn out to have the biological profile of the victim.” The report continued (p. 279), “And it is probable - not necessary, but probable - that during the following act of scrubbing the hands to remove the blood, he/she left the mixed trace consisting of Meredith’s blood and of cells which had been removed by rubbing during the act of washing.”

The Massei motivations report acknowledged that Amanda had no wounds and therefore was not bleeding. It also noted that DNA by itself gives no indication of when it was deposited (see below). In summary Massei thought that the mixed DNA did not necessarily indicate mixed blood, but he believed that the traces were deposited simultaneously, at least partially on the basis of Amanda’s declaring that the bathroom was clean on the afternoon of 1 November. Such a position is problematic in that a clean bathroom does not necessarily imply a DNA-free bathroom. Moreover, there is no reason to rule out Amanda’s depositing the DNA on the morning after the crime in addition to the possibility that she deposited it before the crime.

Does mixed DNA have to be deposited at the same time?In general the presence of DNA almost never gives an indication of how or when it was deposited. The abstract of an article (“DNA profiling of trace DNA recovered from bedding,” Forensic Science International, Volume 159, Issue 1, 25 May 2006, Pages 21-26) on DNA profiling states in part: “The results indicate that the DNA profile of an individual can be obtained from bedding after one night of sleeping in a bed. The DNA profile of the owner of the bed could also be detected in the foreign bed experiments. Since mixed DNA profiles can be obtained from trace DNA on bedding, caution should be exercised when drawing conclusions from DNA profiling results obtained from such samples.” This is a good example of mixed DNA that could not have been deposited simultaneously.

How common is mixed DNA?Head of the US National Institute of Standards and Technology's genetics group, “[John] Butler has reviewed more than 5000 DNA samples from 14 US labs and found that mixing is a common occurrence: 34 per cent of the samples he studied included DNA from two people, while 11 per cent were three or four-person mixtures.” Although some fraction of the two-person samples are from the victim and the perpretrator, it is not reasonable to suppose that this is the case for all of them, let alone the three or four-person mixtures.

Are there other mixed DNA samples in this case?In addition to the mixed DNA of Meredith and Amanda at the girls’ flat, there are also three mixed DNA samples containing Amanda’s and Raffaele’s DNA at his flat. One was found in Sollecito’s bathroom, one was found in his bedroom, and one was found on a pair of rubber gloves. The former two are also luminol-positive, but the identity of the luminol-reactive substance is not known. All three mixed samples are likely to be the result of cohabitation. Amanda’s and Raffaele’s DNA was also found on a cigarette butt at the girls’ flat (p. 193, Massei Report, English translation). The cigarette butt is also interesting in that some of the peaks comprising Amanda’s profile are moderate in intensity, despite possibly being the result of secondary DNA transfer (Amanda does not smoke cigarettes).

Do any of the samples contain DNA from a third party?Sara Gino’s testimony indicated that in sample 177 in Filomena’s room there were alleles besides those of Meredith and Amanda. I have also examined a copy of the electropherogram. In the D19S433 locus, four alleles are marked: 12, 13, 16, and 16.2, but there are unlabeled alleles at 14 and 15 or 15.2. If one acknowledges that it might have been deposited at some other time than the murder, then one must also acknowledge the same possibility for Amanda's DNA.

Could the forensic team have run controls?Besides the issue of how samples were collected in general, the forensic police could have done substrate controls, where they examined areas for DNA that were a few inches away from putative blood stains, as materials scientist Dr. Mark Waterbury suggested. If they had found Amanda’s DNA in some of those locations, it would have been suggestive of innocent DNA deposition.

They could also have performed or cited studies of DNA deposition in bathrooms, if such studies existed already (I am not aware of any). There are a number of ways that such studies could be performed. For instance, one could take blood from person A and place it in person B’s bathroom, then collect DNA samples. If one found mixed DNA from A and B, it would strengthen the hypothesis that Amanda deposited DNA in the normal course of everyday living.

Is there precedent for mixed DNA arising through contamination?Many cases of contamination show a mixture of DNA from the analyst and a potential suspect, as discussed in the previous blog entry. One case, the murder of Jane Mixer, showed contamination from two potential suspects, Gary Leiterman and John Ruelas. However, Ruelas was four years old at the time of the murder and lived in a different city. Therefore, this is probably a case in which both profiles arose from contamination.

Could the way the DNA was sampled have resulted in mixed DNA?The Massei Motivations report (p. 278, English translation) indicates that the defense thought that the mixed traces were meaningless: “All the more so since the samples had been taken using the same blotting paper which had been used for various parts of the bidet and the sink.” Even Colonel Garofano (a strongly pro-prosecution commentator on the case) was dismayed at the way the washbasin trace was collected, noting, “The fact that the sample was collected by wiping both the edge and the plughole is dangerous. You’re likely to find all sorts of stuff in the plughole.” (p. 370, “Darkness Descending”)

Did the police take every precaution to avoid contamination?No, there are several ways in which the work could have been improved. Ms. Stefanoni’s view was that liquid samples are liable to cross-contamination, but dry traces are not. In the English translation of the Massei report (p. 203) it says that Stefanoni “specified” that gloves were changed “every time an object was touched that was particularly soaked with blood, and when it was obvious that the gloves would be soiled;” On pages 204-205 she indicated that the presence of a liquid is necessary to bring about contamination by touch.

Ms. Stefanoni’s view is out of the mainstream. On page 38 of John Butler's textbook “Forensic DNA Typing,” he wrote, “Use clean latex gloves for collecting each item of evidence. Gloves should be changed between handling of different items of evidence.” At Forensic Magazine in the article “Evidence Handling and Collection” Dick Warrington wrote, “Go about collecting evidence. I can’t say enough about avoiding cross contamination. Put on gloves, use gloves, change gloves. Do that every time you touch a piece of evidence. Likewise, use disposable tweezers, scalpels, etc. Change these each time they are used, as well.” Warrington also wrote an article for Forensic Magazine called “DNA Collection and Packaging,” that discussed the use of gloves and tweezers to avoid contamination. Orchid Cellmark’s guidelines state, “Use clean latex gloves for collecting each item of evidence. It is recommended the gloves be changed between the collection of each item of evidence.” If the police handled an item of evidence with Amanda’s DNA then handled an item with Meredith’s DNA, the glove could carry Amanda’s DNA into the other sample.

Are there innocent explanations for the mixed DNA in Filomena’s room?The luminol work that first identified some of the areas that later were shown to contain mixed DNA traces was performed on December 18, 2007. By this time many police personnel had been in the girls’ flat, and many of Meredith’s items had been tossed about. This raises the possibility that the forensic police tracked the genetic material of either Knox or Kercher into Filomena’s room from the hall. The forensic police who were recorded on 18 December wore one-piece tyvek garments but did not appear to have any outer shoe covering. Former FBI agent Steve Moore noted that they did not change shoe covers going from one room to another that that this creates the potential for cross-contamination. This is especially worrisome in that several members of the team are quite close to the dried bloodstains in Meredith’s room, as can be seen in parts 10 and 11 of a series of videos taken on 18 December 2007.

In addition the luminol-positive spots are only presumptive blood; these tested negative by tetramethylbenzidine, a second type of presumptive test, and there is no record of confirmatory blood testing. Therefore, it is open to debate whether or not the luminol-positive substance is even blood. One photograph of the luminol-positive footprints in the hallway also show blue specks on the ruler and on the boot of one of the forensic police officers. It is unclear what the luminol-positive substance was in this case, or whether it could have contaminated other items of evidence.

ConclusionsMixed DNA is commonly observed and is not equivalent to mixed blood. In general DNA samples cannot be dated, and any two profiles within a sample may have been deposited at different times. The mixed DNA in the bathroom may have been created by Meredith's blood falling on Amanda's biological matter that was already there. The chances of this happening might have been lessened if the forensic police had taken a smaller trace with respect to the washbasin, for example. Dirty gloves or dropped swabs (which happened elsewhere) made have mixed DNA during collection. The police or the inhabitants of the flat may have tracked Meredith's blood into Filomena's room. The evidentiary value of these mixed DNA samples is very low.

Good article Chris ! Looks like maybe the experts from Rome may get another shot at making the Prosecution look like a bunch of school kids. If they were so sloppy in MK room why would anything be different here !

There are a few drops of blood on the sink faucet that (to the best of my knowledge) had only Amanda's DNA. Amanda believed that her attempt to pierce her ear(s) caused these droplets. These drops are difficult to see in most photographs, but they do show up under strong light. IIRC there was a bloodstain on Amanda's pillow, but I am not certain whether or not that was tested.

Thanks for the info. I posted questions on the Injustice in Perugia website that you have answered very well albeit indirectly. I referred to virtually the same points in the Massai report that you have quoted yourself. My conclusion was that if it was not possible to ascertain who the blood belonged to in the mixed trace samples, it would commonsense to conclude that the mixed blood theory could not be proven.

After all the controversy about the blood in the cottage I still don’t even know what blood group either of the girls were. I am particularly reassured that that high spikes of DNA can come from saliva, and an accumulation of this from brushing teeth, gargling, and hand washing after toilet use would certainly account for Amanda’s DNA being on the samples. Even if Amanda’s blood was involved, her recent ear piercing and or menstrual blood could be as a perfectly valid reason.

I agree about the Duke lacrosse case. Apparently Manuela Comodi, one of the prosecutors, brought up convictions in the pre-DNA era, which is reminscent of Nifong's comments about convicting on rape charges the good, old-fashioned way. What they ignore in each case is the evidence that should be there, but is not.

The court received an anonymous letter from someone apparently within the forensic police, detailing lack of funds for basic equipment and things like that. I don't believe that the letter has any legal weight, but I suspect it has more than a grain of truth.

I think your implication is correct; the only way to prove or to disprove mixed blood would be to test for antigens (such as the ABO biomolecule) that would be specific both to blood and to the two individuals in questions.

Holding my breath in anticipation of this veridct and thinking how much I appreciate the efforts of so many who have put forth such a sustained and dedicated effort to right the terrible injustice in Perugia.

Thank you very much for your kind words and your continued interest in the case. What a relief that the nightmare is over. However, a commenter named SkewedView at WebSleuths made a point that I think is worth sharing, "While I am very, very happy that AK & RS have been freed, I can't help hurting even more so for the Kerchers at this time. They are now stuck in a catch 22 that will never allow them to find true peace. They must now choose to either keep believing in the prosecutors etc and thus feel that their daughter was cheated out of justice, or they must accept the ruling as a true one, and thus deal with the guilt of having cheered a grave injustice being perpetrated in their loved one's name. I can tell you, it is a horror that nobody should ever have to deal with, and my heart goes out to them." I hope they are able to find peace of mind.

I share your and others' joy in the appeal verdict of the Italian Court.

However, I strongly disagree that the Kerchers "are now stuck in a catch 22 that will never allow them to find true peace." That is nonsense. The man who murdered their daughter was convicted and is in jail serving a sentence for his crime of killing the Kerchers' daughter. That's justice, and that is a hell of a lot more "true peace" than many families of murder victims ever get.

To have two people convicted of a crime that they did not commit and for which there was not a scintilla of proof of their guilt is not justice. "True peace" is not found in seeing innocent people convicted of a crime, regardless of how terrible the pain of the Kercher family must be.

I keep reading observations in reports on this verdict that "lost in all of this drama are the Kerchers."

That's baloney.

"Lost in all of this" is the simple fact that the Italian prosecutors initially charged the wrong persons for this crime based on a completely fabricated theory, and when they actually found the perpetrator of the crime; solid evidence placing him at the scene; and ample forensic evidence of his guilt, the prosecutors allowed their pride and egos to trump any sense and responsibility they might have had to "do justice" and drop the charges against Knox and Sollecito.

The actions of the Italian prosecutors in their treatment of Knox and Sollecito was a travesty of justice and it very nearly cost two demonstrably innocent people 20+ years of their lives in prison.

Lyle Kercher talked about being back to square one today. Recently John Kercher said that there were two knives in the murder (there weren't). I think that the family genuinely believes in the multiple-assailant theory. If the family does come to see the single-assailant theory as correct, they will have to come to terms with Rudy's sixteen year sentence, which may get further reduced for good behavior.

I thought that Comodi's statement was pretty strange since I had always read that there were several different DNAs on the clasp. Reading some of those long ago arguments on the JREF about the clasp and other places, I thought there were maybe 4 or 5 different DNAs on the clasp.

I'd have to say watching the live feed on CNN my heart was in my throat just thinking about what AK and RS must have been thinking. A few moments to decide the next 20 years or longer.

The poor Kerchers...I'm sure they believed everything the prosecutors were telling them- why would you not if you were in their position really. I think it was Meredith's mom who said at the end of the first trial "'If the evidence has been presented then yes you have to agree with that verdict. 'It's difficult to say but at the end of the day you have to go on the evidence because there's nothing else.' " I believe that evidence is why they were acquitted this time.

finally, I would like to hope that some crazy person does not decide to take some sort of "justice" in to their own hands and tries to harm Amanda. Enough lives have been damaged, there is no need for more tragedy.

Chris, I certainly agree with you regarding the misunderstanding of the Kercher family, but the "multiple assassin" theory always was a complete fabrication of the Italian prosecutor whose perfidy to the cause of justice makes Mike Nifong look like an amateur.

The Italian prosecutor's actions in this case are the very reason he stands charged today of similar crimes.

I cannot understand how any reasonable person could possibly believe that three people together at the same time committed a brutal, bloody assault and murder of a woman and yet only one of those individuals left any trace of DNA, bloody footprints or other evidence indicating that they were present at the scene of the crime. And, such evidence from the person who actually did commit the crime was found in abundance.

I cannot possibly imagine how overwhelming the grief must be for the Kercher family, and I hope and pray that they will someday understand that the sole and only person who murdered their daughter is behind bars and no other person escaped justice for their daughter's murder.

Thank you for all of your efforts in helping us understand better the DNA issues. It has been a long haul!

I appreciate your scientific demeanor. IMHO, you presented the facts without taking sides or claiming more than what the science supported. You were careful to distinguish between was known and what was rumor.

Thanks so much for the article. It is great to have an objective analysis of the DNA evidence. I wish the journalists covering the case would take the time to do a little reading of material like yours--it might help them to stop spouting the half-baked half truths or mostly lies that they do spout, stirring the mob hysteria nicely. Thank heavens the appeal went well, and that there apparently are some honorable and intelligent members of the Italian judiciary, among which I do NOT include the prosecutors, who ought to be in jail themselves. What a travesty they perpetrated.

I wish I had been the Observer in the previous comment. Whoever you are, you must have read my mind. I couldn't have said it better.

Mr. Mignini has been a horror to everyone, including the Kerchers. His request for a life sentence and solitary confinement (seemingly with the full support of his fellow prosecutors) for his non-existent case...what is that? And to pull the Kerchers along on this inane mission...maybe it's time for someone to call in an exorcist.

Perhaps Judge Hellman will be able to explain to the Kerchers about the liklihood of a lone assailant in his opinion. That's really who should provide the explanation. Barring that, though, FBI Agents Steve Moore and Friend do quite well.

I have heard Ron Hendry's name mentioned as someone who could explain it to the Kercher family also. Those who know him have good things to say about his kindness as well as his amazing skills at reading a crime scene. Jim Lovering indicated that Ron is strongly of the opinion that this was a single-assailant crime.

RD,

Thank you. I still feel that DNA profiling is the queen of forensics, but there are some limitations that should always be acknowledged.

Randy,

I posted an analysis at JREF on the clasp, but I have not updated my clasp blog entry yet. I will try to do so this weekend. In my view there were multiple profiles, but the Conti-Vecchiotti report should be consulted for more details.

Do you have any comment on the question whether exfoliated skin cells contain DNA? Stefanoni is supposed to have said thy did not in the translated Massei report (I don't have the page reference to hand but I can look it up if required).

I am talking about the human cells which wind up in house dust and I am alluding to the possibility that the bra clasp showed up Raffaele's DNA because it collected dust.

There is a good thread at JREF about skin cells and dust. My present understanding is that when a skin cells goes through the process of keratinization, the nucleus may break down, which leads to degradation of the DNA. However, not all dead skin cells lose their DNA, or else it would be difficult to explain the DNA in dust or in fingerprints. There is a good journal article by Toothman et al. in 2008 on the subject of DNA in household dust that is cited by the Conti-Vecchiotti report. This journal article is mentioned in the JREF thread.

I don't think that household dust is a likely explanation for Sollecito's putative profile on the clasp. He did not spend a great deal of time at the girls' flat. However, he did leave fingerprints on the outside of Meredith's door. If a technician touched the door then collected the clasp, that is a possible mechanism of transfer. There are a couple of mechanisms one can envision involving the towels that Rudi brought into Meredith's room also.

I shall look at the JREF link you helpfully give. I think I also recall from the Conti-Vechiotti report that the locus of the DNA was reported to be on one of the hooks of the clasp, not the cloth which might be a better gatherer of dust.

"Blood was found in seven locations in the small bathroom that Knox shared with Meredith. [192]

• The Door Frame: blood was found on the right, inside door frame containing Meredith’s DNA. [192] • The Light Switch Plate: Meredith’s blood was also found on the light switch. [192] • The Sink: Blood was found in two places. There was dried blood near the faucet that had the DNA of Knox. [192] A streak from the left part of the sink toward the drain containing Meredith’s blood mixed with DNA of Knox.[192] • The Bidet: Meredith’s blood was found mixed with the DNA of Knox.[192] • The Toilet Lid: Meredith’s blood.[192] • Q-tip Box: Meredith’s blood mixed with DNA of Knox.[192] • The Bathmat: Three samples taken from the bathmat yielded Meredith’s blood.[192]

The fatal wound was swabbed in order to obtain the profile of her DNA for comparison with other samples. [190] One of two swabs of her vagina produced genetic material, the DNA of the Y chromosome of Rudy Guede. [189] Samples taken from under her fingernails yielded only her own DNA. The court noted that her finger nails were very short and probably would not inflict significant scratches on an attacker. [190]

Rudy Guede’s Y chromosome was also found mixed with Meredith’s blood on Meredith’s handbag and on the left cuff of her sweatshirt.[192]"

The circumstances seem to suggest that someone cleaned themselves up in the bathroom generally used by Meredith and Amanda which is why there is so much evidence of Merediths blood. However there is no evidence of Guede in this bathroom. Isnt this a little odd?

There is clearly blood on the Bra clasp - dried blood is visible on the clasp. It is from this that Sollecitos DNA was recovered. Who put the blood on the clasp?

The blood on the clasp may or may not be the source of DNA on the clasp, Meredith's or anyone else's. The only thing that Guede did that would potentially have left much DNA in the bathroom is to bleed, and I am not certain that he was bleeding. It would have been nice to be able to test the towels, but they were stored improperly. Guede's blood might have been there.

About Me

I am a biochemist who specializes in the chemical modification of proteins and the synthesis of potential enzyme inhibitors. I am particularly interested in the chemistry of phosphorus and sulfur as it can be applied to biochemical problems.